Two of the most devastating problems in cancer treatment are drug-toxicity, which debilitates patients, and drug-resistance, which is normally countered with even higher drug dosages and thus amplifies the problem of drug-toxicity, often resulting in death. One way to solve the problem of drug-toxicity is to target drugs for delivery only to cancer cells. Many researchers are working to develop antibodies to deliver drugs to targeted cells, and this approach holds promise, but antibodies are not without problems. For example, antibodies often bind to normal tissues, and they also can damage blood vessels (e.g., vascular leak syndrome) and cause dangerous allergic reactions (e.g. anaphylaxis).
Research is also progressing in connection with the use of conjugates of transferrin and doxorubicin, daunomycin, methotrexate, vincristin, 6-mercaptopurine, cytosine arabinoside, cyclophosphamide, and radioiodine as described in U.S. Pat. Nos. 5,108,987; 5,000,935; 4,895,714; and 4,886,780. The inventions described in these patents does not use antibodies. Instead, it uses a protein found in normal human blood. This protein is transferrin, which delivers iron. Normal cells rarely require iron, but cancer cells require large amounts of iron to maintain their pathologically increased rates of metabolism. Because cancer cells require more iron, they have transferrin receptors substantially permanently on their surfaces, whereas normal cells do not. These inventions exploit these receptors by administering anticancer drugs conjugated with transferrin, which delivers the drugs substantially only to the surface of cancer cells.
Drug targeting spares normal cells, requires less drug, and significantly diminishes drug-toxicity. In contrast, when anticancer drugs are administered without being targeted, they kill normal cells as well as cancer cells. They are particularly toxic to cells of the immune system and to the system responsible for blood clotting. Thus, infections and bleeding are principal complications of chemotherapy in cancer patients. These complications require expensive services, hospitalizations, intensive care, and life-support systems, which are uncomfortable and expensive for the patient. These problems are largely preventable by using targeted delivery systems.
The problem of drug-toxicity consumes huge blocks of doctors' and nurses' time, and is responsible for much of the cost of cancer care. For example, it is commonly understood that about 70% of calls to oncologists relate to a problem of drug-toxicity. Today there is no satisfactory way to treat drug-toxicity, except to use less drug. In the absence of targeted delivery the use of less drug is counterintuitive in the case of drug resistant cancers. Targeted delivery allows the use of less drug, because more of the administered drug is delivered specifically to cancer cells rather than being nonspecifically distributed around the body. In this sense, targeted delivery is like shooting with a rifle, while conventional delivery is like shooting with a shotgun. A solution to the problem of drug-toxicity will dramatically transform chemotherapy in cancer patients. It is a purpose of this invention to reduce such adverse effects of chemotherapy.
The problem of drug-resistance is equally as serious as the problem of drug-toxicity. This problem is typified by a patient diagnosed with cancer who is treated and responds with a symptomless remission that lasts many months, and who later sees the cancer returns in a form that no longer responds to any known drug. This scenario of drug-resistance is all too common, Yet today there is no satisfactory solution, except the use of larger amounts of more powerful drugs, that in turn can cause serious drug-toxicity problems, often resulting in death. Thus, a solution to the problem of drug-resistance would significantly diminish the problem of drug-toxicity. A major effort was devoted to the use of P-glycoprotein inhibitors such as verapamil (Ford, Hematol/OncolClin N Am 1995; 9:337), cyclosporine (Bartlett et al, J Clin Oncol 1994; 12:835) and cyclosporine derivatives such as SDZ PSC 833 (Kusunoki et al., Jpn J Cancer Res 1999; 89:1220), and tamoxifen (Pommerenke et al., J Cancer Res Clin Oncol 1994; 120:422), but these approaches have not been clinically satisfactory because they introduced new problems in the pharmacokinetics of chemotherapy (Sikic et al., Cancer Chemother Pharmacol 1997; 40:S13). Other approaches are the design of MDR-reversing drugs (Naito & Tsuruo, Cancer Chemother Pharmacol 1997; 40:S20); the use of P-glycoprotein antisense oligonucleotides (Bertram et al, Anti-Cancer Drugs 1995; 6:124); the use of retrovirus-mediated transfer of anti-MDR ribozymes (Wang et al., Human Gene Therapy, 1999; 10:1185), and the design of chemotherapy drugs that are not removed from cancer cells by MDR or MRP pumps (Mankhetkom et al., Mol Pharmacol 1996; 49:532), but none of this research has provided a solution to the clinical problem of drug resistance (Arceci, Br J Haematol 2000; 110:285). Another approach is to circumvent the MDR/MRP pumps by delivering anti-cancer drugs as conjugates of larger molecules such as albumin (Ohkawa et al., Cancer Res 1993; 53:4238), alpha-fetoprotein (Moskaleva et al., Cell Biol Int 1997; 21:793) and dextran (Fong et al., Anticancer Res 1996; 16:3773). Although these are variously effective at evading experimental drug resistance, they are unproven in patients. Protein-targeted drug delivery can overcome the problem of drug-resistance. Thus, another purpose of the present invention is to resolve the issue of painful and expensive deaths from drug-resistant cancers.
The effectiveness of proteins conjugated with bio-affecting molecules has been demonstrated and is described in the U.S. patents mentioned above. It has been determined; however, that the efficiency of such conjugates in treating stressed cells, such as cancer cells, is reduced by the presence of agglutinated conjugates or by the presence of conjugates of a bio-affecting molecule with protein fragments or with two or three protein molecules and is greatly enhanced when the protein to bio-affecting molecule ratio is closer to 1:1 or 1:2, depending on the bio-affecting molecule. Obtaining conjugates of higher efficiency has, in the past, been a slow, tedious and expensive process that requires separating a fraction of conjugate having the desired average ratio of bio-affecting molecule to protein from a larger sample comprising such molecules conjugated with protein fragments, with a plurality of proteins and proteins conjugated with a plurality of bio-affecting molecules. Using homogeneous protein-drug conjugates in which the protein component carries a predetermined number of bio-affecting molecules can more effectively kill both drug-resistant and drug-sensitive cancer cells. In the past the expense and inefficiency inherent in producing useful conjugates in a useful volume has been a problem for the commercialization of such conjugates and for their widespread use in medicine. There is a need for a substantially homogeneous drug-protein conjugate and for a method of making such a conjugate that is more efficient, more precise and less costly. It is one purpose of this invention to provide such a homogeneous conjugate made by a more efficient method.